CA1340112C - Supression of cell proliferation by decorin - Google Patents
Supression of cell proliferation by decorinInfo
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- CA1340112C CA1340112C CA000604090A CA604090A CA1340112C CA 1340112 C CA1340112 C CA 1340112C CA 000604090 A CA000604090 A CA 000604090A CA 604090 A CA604090 A CA 604090A CA 1340112 C CA1340112 C CA 1340112C
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Abstract
The present invention relates to the proteoglycan Decorin (also known as PG-II or PG-40). The invention provides cells transfected with and expressing a gene coding for Decorin. Spent culture media from such transfected cell cultures can be used to suppress the proliferation of either normal or abnormal cells. Purified Decorin can be used to suppress cell proliferation.
Description
t~ n).~2 SUPPRESSION OF CELL PROLIFERATION BY DECORIN
FIELD OF THE INVENTION
This inventicn relates to cell biology and more specifically to the control of cell proliferation.
BACKGROUND OF THE INVENTION
Under normal ,-ircumstances, cell proliferation is a tightly controlled process; rapid proliferation is needed during embryonal development and tissue regeneration, whereas the proliferation must be halted in the completed tissue.
Cell proliferation appears to be controlled primarily by growth factors. ~ost of the known growth factors are stimulatory, and include epidermal growth factor, platelet-derived growth factor, various interleukins and colony-stimulating factors. A few negative regulators of cell proliferation are a~lso known. Transforming growth factor beta is a multifunctional factor that inhibits the growth of some cell types, but can also stimulate proliferation. Other growth inhibitors include various interferons and a growth inhibitory role has also been ascribed to heparin, heparan sulfate and their fragments.
A less well understood mechanism of growth control relates to the close apposition of cells. Normal cells stop growing when they make contact with one another. This phenomenon, commonly known as contact inhibition of growth, is of obvious importance for the formation of orderly tissue structure.
A number of important pathological conditions result from abnormal cell proliferation. The foremost of such ~C
conditions is, of course, cancer. Other ~1i~q~es with a prolir .ali~e component include ,l.~.. 1oid ~llllilis with its o~_r~luwlh of the synovial tissue, glomerulo~ilis, in which the ...*~ iq1 cells proliferate, and athero~lerosis, in which the abl~l,nally prolir~,.alhlg cells are smooth musc1e cells.
It is obvious from these examples that there is a great need to develop new methods for controlling cell proliferation. The present invention addresses this need and provides other related advantages as well.
SUMMARY OF THE INVENTION
The present invention relates to the proteoglycan Decorin (also known as PG-II or PG 40). The invention provides cells ll~llsÇ~d with and eA~lessi~g the gene coding for Decorin, and recolu~ l Decorin produced lh~ . Spent culture media from such lla~çec~l cell cultures can be used to S~lppl~SS the proliferation of either normal or abnnrm~1 cells. Moreover, ~ulirled Decorin can be used to ~upplei:,S cell proliferation.
Thus the present invention provides, in one e.nbodi"..,.ll euk~otic cells ll~r~dwith decorin cDNA, as well as the use of such cells for the production of recolll~inant decorin.
In another embodiment the invention provides a method of ~u~l~ssing proliferation of cells by c0~1; ct;ue said cells with spent culture media from decorin-producing cells, or from cells Ll~Çecled with decorin cDNA.
The invention provides a method of ~upplessillg proliferation of cells by cont~~tin~
said cells with decorin which is free of animal plOIeh~S notmq11y associal~d with it.
In another embc~limPnt, the present invention provides the use of decorin for the plepardtiûn of a ."~dir~-..Pnt for the ll~ of ~li~a~s with a proliferative component, wl~.ein the di~ase may be, for example, .h.-....~toid ~Ihlilis, glomerulo~ep~ilis, or atherosclerosis. In another aspect the invention provides a u~ of decorin for the pl~palalion of a m~ir~ for hl~lue~Y~ the type of and/or quantity of extra~ r matrLc components.
, 1~ lOIl2 BRIEF DESCRIPIION OF THE DRAWINGS
FIGURE 1 shows the ~ ,ssion of Decorin in unamplified and amplified transfectants.
FIGURE 2 is a radiogram ~ w~ng the e}.i)lession of Decorin core protein in CHO
cells.
FIGURE 3, panels A B, C and D, shows morphological changes caused by e*,ies~ion of Decorin in CHC) cells.
FIGURE 4 is a graph showing the growth of Decorin-~r~s~mg and control CHO
cells in culture.
FIGURE 5, panels A and B, are photomicrographs showing the effect of spent culture media on the morphology of CHO cells.
FIGURE 6, panels A and B, are photomicrographs showing the effect of spent culture media on the morphology of Harvey ras gene-transformed NIH 3T3 cells.
FIGURE 7 shows the elution pattern from DEAE-Sepharose of spent culture media from Decorin-e~lessillg cell lLne, clone 61.
DETAILE~ DESCRIPTION OF THE INVENTION
Proteoglycans are proteins that carry one or more glyco~minoglycan chains. The known proteoglycans carry out a variety of functions and are found in a variety of cellular locations. Many of them, however, are C~J111~>01~.I1S of extracellular matrix, where they participate in the assembly of cells to the matrix and affect the ~tt3~ mf~nt of cells to the matrix.
Decorin, also kn~wn ;lS PG-II or PG~0, is a small proteoglycan produced by fibroblasts. Its core protein has a molecular weight of about 40,000 daltons. The core has been sequenr~d (Krusius and Rllo~lqhti, Proc. Natl. Acad. Sci. USA 83:7683 (1986); Day et al. BiorllP-m J. 248:801 (1987)), and it is known to carry a single glycosqrninoglycan chain of a cl1ol~loili~ sulfate/dermatan sulfate type (P.al~ol~, et al., J. Biol. Chem 258:15101 (1983)).
The only previou~ly known function for Decorin is its binding to type I and type II collagen and the effect it has on the fibril formation by this collagen (Vogel, et al., Biochem. J. 223:587 (1984)).
A molecular biological ~tudy of Decorin has now led to unexpected observations on its role in the control of cell proliferation, and these observations form the basis of this invention.
Decorin cDNA is transfected into cells, such as Chinese hamster o~ary ~CH0) cells, preferably those which are di~ydrofolate reductase tdhfr)-negative, although other cells such as 3T3 and COS cells can also be used. Such transfection is accomplished by methods well-known in the art. The transfected cells are then grown in culture.
Chinese hamster ovary (CH~) cells into which human Decorin cDNA was transfected and which express the proteoglycan from this cDffA appear more adhesive to the substratum than the original cells. Moreover, the growth of the cells that expressed Decorin from the cDNA was suppressed and they grew to a lower ~aturation density than the various control cells. These contro~s included cells transfected with a construct expressing t~e core protein of Decorin and amplified to the 6ame degree as the Decorin expressing cells.
These cells were similar to the original CH0 cells. The magn$tude of the growth and adhesion changes was proportional to the amount of Decorin produced.
Moreover, changes in the adhesion and the saturation density could be reproduced with the ~pent culture media of the cells expressing the recombinant Decorin and with the Decorin isolated and purified from such culture media. These findings indicate that Decorin plays a previously unsuspected role in the control of cell proliferation, and that it can be used to modulate cell proliferation. The effect seen with oncogene-transformed 3T3 cells suggests that this invention may be useful in the treatment of proliferative diseases.
As used herein "Decorin" referes to a proteoglycan having the structural characteristics attributed to it in Krusius and Ruoslahti, supra, and which suppresses cell proliferat~on as determined by the method of Example III.
Human fibro~last Decor~n has substantially the amino acid sequence presented in Krusius and Ruos~ahti supra, figure 2.
"Decorin" refers both to the native composition and to modifications thereof which retain the functional characteristics.
As used herein, the term "substantially purified Decorin" refers to the level of purity obtained by the experimental procedure de~cribed in Example IV.
The recom~inant Decorin of the invention has a structure corresponding substantially to that of the native proteoglycan. It is understood however that limited modifications may be made however without destroying the Decorin activity.
EXAMPLE I
~ XP~ STON OF DR~ORIN ~ DRCORTN CORE PROTEIN
The 1.8 kb full-length Decorin cDNA described in Rrusius and Ruoslahti, Proc. Natl. Acad. Sci. USA 83:7683 (1986), wa~
used for the con~truction of Decorin expression ~ectors. For the expression of Decorin core protein, a mutagenized cDNA in which the fourth codon, TCT coding for serine, was changed to ~ X
i f~ ~ ~ 2 ACT coding for threonine was engineered by site-directed mutagenesis according to the method of Kunkel, Proc Natl Acad Sci USA 82 488 ~1985) The mammalian ~xpre~sion ~ectors -pSV2-Decorin and pS~2-Decorin/CP (core prot-in) were con-truct~d by ligating th- Decorin cDNA or the ~utagenized D-corin cDNA into 3~ kb ~indlII - a~ HI fragment of pSV2 (Mulliqan and ~erg, 5cience 209 1423 (1980) re~pectively Dihydrofolate r-ducta~e (dhfr)-negative CHo ccll~ (CH0-DG44) were cotransfected with pSV2-Decorin or pSV2-Decorin/CP ~nd pSV2dhfr by the calcium p~osphate precipitation method (Graha~, F and Van der E~, Virology S2 ~56 (1973) The transfected cells were cultur d in nucleosidc-minus alpha-modlfied ~inimal e~ential ~edium l~-MEM, GIBC0, Long I~land) ~uppl-mented with 9~ dialyzed f-tal calf ~erum, 2 ~M gluta~ine, 100 units/~l penicillin and 100 ~g/ml ~treptomycin Colonies arieing from transf-cted cell~ were picked using cloning cylinder~ pand~d and checked for the expression of Decorin by iDmunoprecipitation from 35S04-labeled culture ~upernatant~ Clones expre~sing a~ubstantial amount of Decorin were then ~ub~ected to gene amplification by ~tepwise increasing c6n_ entration of ~ethotrexate (MTX, ~aufman and Sharp, J Mol Biol 159 601 (1982) up to 0 64 ~M ~11 the a~plifiea cell line~ were cloned either by limiting dilut~o~ or ~y picking ~ngle MTX resistant colonie~ 8toc~ culture~ of the~e establi~hed cell lines were kept in MTX-containing medium Before use in exp-rlment~, cell~ were 6ubcultured in MTX-minus medium from ~tock cultures and pas~ed at least once in this ~edium to eliminate the po6~ible MTX eff-ct~ Controls were tran~fected only with pSV2dhfr and treatcd exactly as experimental cells thereafter Metabolic labeling of the cell~ with 35S04 or 3H-leucine and i~munoprecipitation was 1 i 2 performed as described 8rennan et al., J. Biol. Chem 259:13742 (1984).
Figure 1 shows the expression of ~ecorin in unamplified and amplified transfectants, by using fluorography of SDS-7%-polyacrylamide gel electrophoresis under reducing conditions.
(A) 35SO4-labeled culture supernatants immunoprecipitated with rabbit antipeptide antiserum prepared against the NH2-- terminus of human Decorin (Krusius and Ruoslahti, supra.).
(B) Total 35S04-labeled products secreted into culture medium. (C) Total 3H-leucine labeled products secreted into culture medium. Lane 1: control transfectant A, an unamplified clone transfected with pSY2dhfr; lane 2: control transfectant C, a clone amplified to 0.64 ~M MTX resistance from control transfectant A; lane 3: clone 1, an unamplified primary transfectant expressing 0.2 pg/cell/day of Decorin;
lane 4: clone 31, a clone amplified to 0.32 ~M MTX
resistance and expressing 4 pg/cell/day of Decorin; lane 5:
clone 61, a clone amplified to 0.64 ~M MTX resistance and expressing 25 pg/cell/day of Decorin.
Figure 2 shows expression of Decorin core protein in CHO
cells. Lanes ~ and 2: 3~-leucine-labeled culture supernatants were immunoprecipitated as described in Figure 1. Lanes 3 and 4: Total 3H-leucine-labeled products secreted into culture medium. Lanes 1 and 3: CHO cells transfected with pSV2-Decorin/CP. Lanes 2 -and 4: Control CHO cells transfected with pSV2dhfr.
EXAMPLE II
OUANTITATION OF CELL SPREADING AND SATURATION DENSITY
The cell lines of Example I were plated in 24 well plates in MTX-minus culture medium at a density of 3x105 cells per well. After 24 hours, medium was replaced (0.3 ml _ 7 r ~, per well) and cells were incubated another 24 hours Concentration of Decorin in these culture ~upernatants was determined by competitive ELISA (Engvall, Neth Enzymol 70 419 (1980) Briefly, ~ ~ixture of culture ~upernatant ~nd r~bbit ~ntipeptide antlbody aga~nst ~ corin ~s ~c~bated ln the ~ell~ of ~icrotiter plat-~ co~ted with D-corin purified from huo~n f-t~ ubran~ t ~ upra ) ~he ~mount of ~ntibody bound to the w lls ~ d-ter~ined by alk~line-pho~ph~t~-~ oo~,~ugated go~t anti-rabbit TgG as ~ ~econd ~ntibody V~rious concentr~tion~ of purifiQd D-corin were u~ed to gener~t- a ctandard ¢urve The cell~ were counted by hemocytometer at th~ end of the 24 hour incubation As shown in Table I, cells transfected with the Decorin gene exhibited a l~rger area of spreading than did control cells W~ere Decorin expression was amplified, area of spreading increased with increasing expression Also 6hown in Table I are the saturation densities of the Decorin-expressing and control cells In order to determine the saturation densities, cells (1 2x105) were plated in a 60 ~m culture dish in M~X-Dinus cu~ture medium After 6 hours, cells were fixed with 3* paraformaldehyde and stained with tolu~dine blue Quantitative evaluation of spreading was performed ~y aeas~ring the surface area of the cells with a ~urface integration progra~ of an image analyzer (Olympus) Nonspread cells were excluded from the measurement The mean and standard deviation of values from 50 cells are shown .1~ 1o ll2 TABLE I
PRODUCTION OF DECORIN AND SPREADING OF TRANSFECTANTS
MTX Decorin Saturation Clone Transfection Resistance Prod. Spread AreaDensity (~M) (~g per 106 (~M2/cell) (x 10-5) cells/day) control pSV2dhfr 0 0 2725 + 627 10.8 + 1.2 line A
10 control pSV2dhfr 0.32 0 2585 + 69310.4 + 2.5 line B
control pSV2dhfr 0.64 0 2659 + 58610.6 + 1.8 line C
clone 1 pSV2-decorin 0 0.2 3368 + 8429.g + 1.6 + pSV2dhfr clone 31 pSV2-decorin 0.32 4 4759 + 8987.3 + 0.2 + pSV2dhfr clone 33 pSV2-decorin 0.32 11 5554 + 10025.2 + 0.2 + pSV2dhfr 20 clone 66 pSV2-decorin 0.64 14 5482 + 1382 4.9 + 0.3 + pSV2dhfr clone 61 pSV2-decorin 0.64 25 6472 + 11474.4 + 0.4 + pSV2dhfr EXAMPLE III
ANALYSIS OF THE EFFECT OF SPENT CULTURE MEDIA
The effect of spent culture media on the morphology of CHO cells and Harvey ras gene-transformed NIH 3T3 cells was determined by plating CHO cells in 35 mm dishes at a density of 2x105 cells/dish in two-day spent media from clone 61 containing approximately 20 ~g/ml of Decorin and in similar media from control cell line C containing no Decorin and 13'~112 cultured, the cell lines being those described in Example I.
Figure 5 shows the morphology of the CHO cells after this treatment and Figure 6 ~hows the morphology of the treated oncogene-transformed 3T3 cells. As can be ~een, the spent culture medium from the Decorin-expressing cell line, clone 31, induced a morphology similar to that observed in the Decorin-expressing cells themselves. The oncogene-transforDed 3T3 cells treated in this manner assume a morphology closely ~imilar to that of normal cells. This morphology is often referred to as "contact inhibited morphology~ and it is considered to be indicative of normal growth control. In accordance with this phenomenon, fewer cells were seen in these cultures compared to the control-media treated cultures. These results indicate that the culture media from the cell lines expressing Decorin reproduces the morphological and growth inhibiting effects seen in the recombinant Decorin-expressing cells themselves.
EXAMPLE IV
PURIFICATION OF DECORIN FROM SPENT CULTURE MEDIA
Clone 61 cells were grown to 90% confluence in 8-175 cm2 culture flasks in nucleosidle ~inus ~-MEM supplemented with 9%
dialyzed fetal calf serum, 2 DM glutamine, 100 units/ml penicillin and 100 ~g/ml streptomycin. At 90% confluence culture media was changed to 25 ml per flask of nucleoside-free ~-MEM ~upplemented with 6% dialyzed fetal calf serum which had been passed ~hrough a DEAE Sepharose*Fast Flow column (Pharmacial equilibrated with 0.25 M NaCl in 0.05 M
phosphate buffer, pH 7.4. Cells were cultured for 3 days, spent media was collected and immediately made to 0.5 mM
phenylmethylsulfonyl fluoride, 1 ~g/ml pepstatin, 0.04 mg/ml aprotinin and 5 mM EDTA.
* Trademark A
1~ ~;0,11~
Four hundred milliliters of the spent media were first passed through gelatin-Sepharose to remove fibronectin and materials which would bind to Sepharose. The flow-through fraction was then mixed with DEAE-Sepharos~ preequilibriated in 50 mM Tris/NCl, pH 7.4, plu~ 0.2 M NaCl and batch absorbed overnight at 4- C with gentle miYing. The slurry was poured into a 1.6 cm x 24 cm column, washed extensively with 50 ~M
Tris/HCl, pH 7.4, containing 0.2 M NaCl and eluted with 0.2 M
- 0.8 ~ linear gradient of NaCl in 50 mM Tris/HCl, pH 7.4.
~ecorin concentration was determined by competitive ELISA as described above.
Figure 7 shows the elution pattern in DEAE-Sepharose Fast Flow. As can ~e seen, Decorin separates from the bulk of the protein present in the media and can be recovered in substantially pure form from the fractions showing the highest iDune reactivity.
Although the invention has been described with reference to the presently-preferred e~bodiment, it should be understood that various modifications can be made without departing from the spirit of the invention. Accordingly, the invention is limited only by the following claims.
* Trademark
FIELD OF THE INVENTION
This inventicn relates to cell biology and more specifically to the control of cell proliferation.
BACKGROUND OF THE INVENTION
Under normal ,-ircumstances, cell proliferation is a tightly controlled process; rapid proliferation is needed during embryonal development and tissue regeneration, whereas the proliferation must be halted in the completed tissue.
Cell proliferation appears to be controlled primarily by growth factors. ~ost of the known growth factors are stimulatory, and include epidermal growth factor, platelet-derived growth factor, various interleukins and colony-stimulating factors. A few negative regulators of cell proliferation are a~lso known. Transforming growth factor beta is a multifunctional factor that inhibits the growth of some cell types, but can also stimulate proliferation. Other growth inhibitors include various interferons and a growth inhibitory role has also been ascribed to heparin, heparan sulfate and their fragments.
A less well understood mechanism of growth control relates to the close apposition of cells. Normal cells stop growing when they make contact with one another. This phenomenon, commonly known as contact inhibition of growth, is of obvious importance for the formation of orderly tissue structure.
A number of important pathological conditions result from abnormal cell proliferation. The foremost of such ~C
conditions is, of course, cancer. Other ~1i~q~es with a prolir .ali~e component include ,l.~.. 1oid ~llllilis with its o~_r~luwlh of the synovial tissue, glomerulo~ilis, in which the ...*~ iq1 cells proliferate, and athero~lerosis, in which the abl~l,nally prolir~,.alhlg cells are smooth musc1e cells.
It is obvious from these examples that there is a great need to develop new methods for controlling cell proliferation. The present invention addresses this need and provides other related advantages as well.
SUMMARY OF THE INVENTION
The present invention relates to the proteoglycan Decorin (also known as PG-II or PG 40). The invention provides cells ll~llsÇ~d with and eA~lessi~g the gene coding for Decorin, and recolu~ l Decorin produced lh~ . Spent culture media from such lla~çec~l cell cultures can be used to S~lppl~SS the proliferation of either normal or abnnrm~1 cells. Moreover, ~ulirled Decorin can be used to ~upplei:,S cell proliferation.
Thus the present invention provides, in one e.nbodi"..,.ll euk~otic cells ll~r~dwith decorin cDNA, as well as the use of such cells for the production of recolll~inant decorin.
In another embodiment the invention provides a method of ~u~l~ssing proliferation of cells by c0~1; ct;ue said cells with spent culture media from decorin-producing cells, or from cells Ll~Çecled with decorin cDNA.
The invention provides a method of ~upplessillg proliferation of cells by cont~~tin~
said cells with decorin which is free of animal plOIeh~S notmq11y associal~d with it.
In another embc~limPnt, the present invention provides the use of decorin for the plepardtiûn of a ."~dir~-..Pnt for the ll~ of ~li~a~s with a proliferative component, wl~.ein the di~ase may be, for example, .h.-....~toid ~Ihlilis, glomerulo~ep~ilis, or atherosclerosis. In another aspect the invention provides a u~ of decorin for the pl~palalion of a m~ir~ for hl~lue~Y~ the type of and/or quantity of extra~ r matrLc components.
, 1~ lOIl2 BRIEF DESCRIPIION OF THE DRAWINGS
FIGURE 1 shows the ~ ,ssion of Decorin in unamplified and amplified transfectants.
FIGURE 2 is a radiogram ~ w~ng the e}.i)lession of Decorin core protein in CHO
cells.
FIGURE 3, panels A B, C and D, shows morphological changes caused by e*,ies~ion of Decorin in CHC) cells.
FIGURE 4 is a graph showing the growth of Decorin-~r~s~mg and control CHO
cells in culture.
FIGURE 5, panels A and B, are photomicrographs showing the effect of spent culture media on the morphology of CHO cells.
FIGURE 6, panels A and B, are photomicrographs showing the effect of spent culture media on the morphology of Harvey ras gene-transformed NIH 3T3 cells.
FIGURE 7 shows the elution pattern from DEAE-Sepharose of spent culture media from Decorin-e~lessillg cell lLne, clone 61.
DETAILE~ DESCRIPTION OF THE INVENTION
Proteoglycans are proteins that carry one or more glyco~minoglycan chains. The known proteoglycans carry out a variety of functions and are found in a variety of cellular locations. Many of them, however, are C~J111~>01~.I1S of extracellular matrix, where they participate in the assembly of cells to the matrix and affect the ~tt3~ mf~nt of cells to the matrix.
Decorin, also kn~wn ;lS PG-II or PG~0, is a small proteoglycan produced by fibroblasts. Its core protein has a molecular weight of about 40,000 daltons. The core has been sequenr~d (Krusius and Rllo~lqhti, Proc. Natl. Acad. Sci. USA 83:7683 (1986); Day et al. BiorllP-m J. 248:801 (1987)), and it is known to carry a single glycosqrninoglycan chain of a cl1ol~loili~ sulfate/dermatan sulfate type (P.al~ol~, et al., J. Biol. Chem 258:15101 (1983)).
The only previou~ly known function for Decorin is its binding to type I and type II collagen and the effect it has on the fibril formation by this collagen (Vogel, et al., Biochem. J. 223:587 (1984)).
A molecular biological ~tudy of Decorin has now led to unexpected observations on its role in the control of cell proliferation, and these observations form the basis of this invention.
Decorin cDNA is transfected into cells, such as Chinese hamster o~ary ~CH0) cells, preferably those which are di~ydrofolate reductase tdhfr)-negative, although other cells such as 3T3 and COS cells can also be used. Such transfection is accomplished by methods well-known in the art. The transfected cells are then grown in culture.
Chinese hamster ovary (CH~) cells into which human Decorin cDNA was transfected and which express the proteoglycan from this cDffA appear more adhesive to the substratum than the original cells. Moreover, the growth of the cells that expressed Decorin from the cDNA was suppressed and they grew to a lower ~aturation density than the various control cells. These contro~s included cells transfected with a construct expressing t~e core protein of Decorin and amplified to the 6ame degree as the Decorin expressing cells.
These cells were similar to the original CH0 cells. The magn$tude of the growth and adhesion changes was proportional to the amount of Decorin produced.
Moreover, changes in the adhesion and the saturation density could be reproduced with the ~pent culture media of the cells expressing the recombinant Decorin and with the Decorin isolated and purified from such culture media. These findings indicate that Decorin plays a previously unsuspected role in the control of cell proliferation, and that it can be used to modulate cell proliferation. The effect seen with oncogene-transformed 3T3 cells suggests that this invention may be useful in the treatment of proliferative diseases.
As used herein "Decorin" referes to a proteoglycan having the structural characteristics attributed to it in Krusius and Ruoslahti, supra, and which suppresses cell proliferat~on as determined by the method of Example III.
Human fibro~last Decor~n has substantially the amino acid sequence presented in Krusius and Ruos~ahti supra, figure 2.
"Decorin" refers both to the native composition and to modifications thereof which retain the functional characteristics.
As used herein, the term "substantially purified Decorin" refers to the level of purity obtained by the experimental procedure de~cribed in Example IV.
The recom~inant Decorin of the invention has a structure corresponding substantially to that of the native proteoglycan. It is understood however that limited modifications may be made however without destroying the Decorin activity.
EXAMPLE I
~ XP~ STON OF DR~ORIN ~ DRCORTN CORE PROTEIN
The 1.8 kb full-length Decorin cDNA described in Rrusius and Ruoslahti, Proc. Natl. Acad. Sci. USA 83:7683 (1986), wa~
used for the con~truction of Decorin expression ~ectors. For the expression of Decorin core protein, a mutagenized cDNA in which the fourth codon, TCT coding for serine, was changed to ~ X
i f~ ~ ~ 2 ACT coding for threonine was engineered by site-directed mutagenesis according to the method of Kunkel, Proc Natl Acad Sci USA 82 488 ~1985) The mammalian ~xpre~sion ~ectors -pSV2-Decorin and pS~2-Decorin/CP (core prot-in) were con-truct~d by ligating th- Decorin cDNA or the ~utagenized D-corin cDNA into 3~ kb ~indlII - a~ HI fragment of pSV2 (Mulliqan and ~erg, 5cience 209 1423 (1980) re~pectively Dihydrofolate r-ducta~e (dhfr)-negative CHo ccll~ (CH0-DG44) were cotransfected with pSV2-Decorin or pSV2-Decorin/CP ~nd pSV2dhfr by the calcium p~osphate precipitation method (Graha~, F and Van der E~, Virology S2 ~56 (1973) The transfected cells were cultur d in nucleosidc-minus alpha-modlfied ~inimal e~ential ~edium l~-MEM, GIBC0, Long I~land) ~uppl-mented with 9~ dialyzed f-tal calf ~erum, 2 ~M gluta~ine, 100 units/~l penicillin and 100 ~g/ml ~treptomycin Colonies arieing from transf-cted cell~ were picked using cloning cylinder~ pand~d and checked for the expression of Decorin by iDmunoprecipitation from 35S04-labeled culture ~upernatant~ Clones expre~sing a~ubstantial amount of Decorin were then ~ub~ected to gene amplification by ~tepwise increasing c6n_ entration of ~ethotrexate (MTX, ~aufman and Sharp, J Mol Biol 159 601 (1982) up to 0 64 ~M ~11 the a~plifiea cell line~ were cloned either by limiting dilut~o~ or ~y picking ~ngle MTX resistant colonie~ 8toc~ culture~ of the~e establi~hed cell lines were kept in MTX-containing medium Before use in exp-rlment~, cell~ were 6ubcultured in MTX-minus medium from ~tock cultures and pas~ed at least once in this ~edium to eliminate the po6~ible MTX eff-ct~ Controls were tran~fected only with pSV2dhfr and treatcd exactly as experimental cells thereafter Metabolic labeling of the cell~ with 35S04 or 3H-leucine and i~munoprecipitation was 1 i 2 performed as described 8rennan et al., J. Biol. Chem 259:13742 (1984).
Figure 1 shows the expression of ~ecorin in unamplified and amplified transfectants, by using fluorography of SDS-7%-polyacrylamide gel electrophoresis under reducing conditions.
(A) 35SO4-labeled culture supernatants immunoprecipitated with rabbit antipeptide antiserum prepared against the NH2-- terminus of human Decorin (Krusius and Ruoslahti, supra.).
(B) Total 35S04-labeled products secreted into culture medium. (C) Total 3H-leucine labeled products secreted into culture medium. Lane 1: control transfectant A, an unamplified clone transfected with pSY2dhfr; lane 2: control transfectant C, a clone amplified to 0.64 ~M MTX resistance from control transfectant A; lane 3: clone 1, an unamplified primary transfectant expressing 0.2 pg/cell/day of Decorin;
lane 4: clone 31, a clone amplified to 0.32 ~M MTX
resistance and expressing 4 pg/cell/day of Decorin; lane 5:
clone 61, a clone amplified to 0.64 ~M MTX resistance and expressing 25 pg/cell/day of Decorin.
Figure 2 shows expression of Decorin core protein in CHO
cells. Lanes ~ and 2: 3~-leucine-labeled culture supernatants were immunoprecipitated as described in Figure 1. Lanes 3 and 4: Total 3H-leucine-labeled products secreted into culture medium. Lanes 1 and 3: CHO cells transfected with pSV2-Decorin/CP. Lanes 2 -and 4: Control CHO cells transfected with pSV2dhfr.
EXAMPLE II
OUANTITATION OF CELL SPREADING AND SATURATION DENSITY
The cell lines of Example I were plated in 24 well plates in MTX-minus culture medium at a density of 3x105 cells per well. After 24 hours, medium was replaced (0.3 ml _ 7 r ~, per well) and cells were incubated another 24 hours Concentration of Decorin in these culture ~upernatants was determined by competitive ELISA (Engvall, Neth Enzymol 70 419 (1980) Briefly, ~ ~ixture of culture ~upernatant ~nd r~bbit ~ntipeptide antlbody aga~nst ~ corin ~s ~c~bated ln the ~ell~ of ~icrotiter plat-~ co~ted with D-corin purified from huo~n f-t~ ubran~ t ~ upra ) ~he ~mount of ~ntibody bound to the w lls ~ d-ter~ined by alk~line-pho~ph~t~-~ oo~,~ugated go~t anti-rabbit TgG as ~ ~econd ~ntibody V~rious concentr~tion~ of purifiQd D-corin were u~ed to gener~t- a ctandard ¢urve The cell~ were counted by hemocytometer at th~ end of the 24 hour incubation As shown in Table I, cells transfected with the Decorin gene exhibited a l~rger area of spreading than did control cells W~ere Decorin expression was amplified, area of spreading increased with increasing expression Also 6hown in Table I are the saturation densities of the Decorin-expressing and control cells In order to determine the saturation densities, cells (1 2x105) were plated in a 60 ~m culture dish in M~X-Dinus cu~ture medium After 6 hours, cells were fixed with 3* paraformaldehyde and stained with tolu~dine blue Quantitative evaluation of spreading was performed ~y aeas~ring the surface area of the cells with a ~urface integration progra~ of an image analyzer (Olympus) Nonspread cells were excluded from the measurement The mean and standard deviation of values from 50 cells are shown .1~ 1o ll2 TABLE I
PRODUCTION OF DECORIN AND SPREADING OF TRANSFECTANTS
MTX Decorin Saturation Clone Transfection Resistance Prod. Spread AreaDensity (~M) (~g per 106 (~M2/cell) (x 10-5) cells/day) control pSV2dhfr 0 0 2725 + 627 10.8 + 1.2 line A
10 control pSV2dhfr 0.32 0 2585 + 69310.4 + 2.5 line B
control pSV2dhfr 0.64 0 2659 + 58610.6 + 1.8 line C
clone 1 pSV2-decorin 0 0.2 3368 + 8429.g + 1.6 + pSV2dhfr clone 31 pSV2-decorin 0.32 4 4759 + 8987.3 + 0.2 + pSV2dhfr clone 33 pSV2-decorin 0.32 11 5554 + 10025.2 + 0.2 + pSV2dhfr 20 clone 66 pSV2-decorin 0.64 14 5482 + 1382 4.9 + 0.3 + pSV2dhfr clone 61 pSV2-decorin 0.64 25 6472 + 11474.4 + 0.4 + pSV2dhfr EXAMPLE III
ANALYSIS OF THE EFFECT OF SPENT CULTURE MEDIA
The effect of spent culture media on the morphology of CHO cells and Harvey ras gene-transformed NIH 3T3 cells was determined by plating CHO cells in 35 mm dishes at a density of 2x105 cells/dish in two-day spent media from clone 61 containing approximately 20 ~g/ml of Decorin and in similar media from control cell line C containing no Decorin and 13'~112 cultured, the cell lines being those described in Example I.
Figure 5 shows the morphology of the CHO cells after this treatment and Figure 6 ~hows the morphology of the treated oncogene-transformed 3T3 cells. As can be ~een, the spent culture medium from the Decorin-expressing cell line, clone 31, induced a morphology similar to that observed in the Decorin-expressing cells themselves. The oncogene-transforDed 3T3 cells treated in this manner assume a morphology closely ~imilar to that of normal cells. This morphology is often referred to as "contact inhibited morphology~ and it is considered to be indicative of normal growth control. In accordance with this phenomenon, fewer cells were seen in these cultures compared to the control-media treated cultures. These results indicate that the culture media from the cell lines expressing Decorin reproduces the morphological and growth inhibiting effects seen in the recombinant Decorin-expressing cells themselves.
EXAMPLE IV
PURIFICATION OF DECORIN FROM SPENT CULTURE MEDIA
Clone 61 cells were grown to 90% confluence in 8-175 cm2 culture flasks in nucleosidle ~inus ~-MEM supplemented with 9%
dialyzed fetal calf serum, 2 DM glutamine, 100 units/ml penicillin and 100 ~g/ml streptomycin. At 90% confluence culture media was changed to 25 ml per flask of nucleoside-free ~-MEM ~upplemented with 6% dialyzed fetal calf serum which had been passed ~hrough a DEAE Sepharose*Fast Flow column (Pharmacial equilibrated with 0.25 M NaCl in 0.05 M
phosphate buffer, pH 7.4. Cells were cultured for 3 days, spent media was collected and immediately made to 0.5 mM
phenylmethylsulfonyl fluoride, 1 ~g/ml pepstatin, 0.04 mg/ml aprotinin and 5 mM EDTA.
* Trademark A
1~ ~;0,11~
Four hundred milliliters of the spent media were first passed through gelatin-Sepharose to remove fibronectin and materials which would bind to Sepharose. The flow-through fraction was then mixed with DEAE-Sepharos~ preequilibriated in 50 mM Tris/NCl, pH 7.4, plu~ 0.2 M NaCl and batch absorbed overnight at 4- C with gentle miYing. The slurry was poured into a 1.6 cm x 24 cm column, washed extensively with 50 ~M
Tris/HCl, pH 7.4, containing 0.2 M NaCl and eluted with 0.2 M
- 0.8 ~ linear gradient of NaCl in 50 mM Tris/HCl, pH 7.4.
~ecorin concentration was determined by competitive ELISA as described above.
Figure 7 shows the elution pattern in DEAE-Sepharose Fast Flow. As can ~e seen, Decorin separates from the bulk of the protein present in the media and can be recovered in substantially pure form from the fractions showing the highest iDune reactivity.
Although the invention has been described with reference to the presently-preferred e~bodiment, it should be understood that various modifications can be made without departing from the spirit of the invention. Accordingly, the invention is limited only by the following claims.
* Trademark
Claims (10)
1. Eukaryotic cells transfected with decorin cDNA.
2. The use of cells according to claim 1 for the production of recombinant decorin.
3. The use of claim 2, wherein the recombinant decorin is free of animal proteins normally associated with it.
4. A method of suppressing proliferation of cells by contacting said cells with spent culture media from decorin-producing cells.
5. A method of suppressing proliferation of cells by contacting said cells with spent culture media from cells transfected with decorin cDNA.
6. A method of suppressing proliferation of cells by contacting said cells with decorin free of animal proteins normally associated with it.
7. The method of claim 6 wherein said decorin is produced by recombinant means.
8. The use of decorin for the preparation of a medicament for the treatment of diseases with a proliferative component.
9. The use of claim 8 wherein the disease is rheumatoid arthritis, glomerulonephritis, or atherosclerosis.
10. The use of decorin for the preparation of a medicament for influencing the type of and/or quantity of extracellular matrix components.
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| US6509314B1 (en) * | 1988-06-28 | 2003-01-21 | The Burnham Institute | Methods of preventing or reducing scarring with decorin or biglycan |
| US5583103A (en) * | 1988-06-28 | 1996-12-10 | La Jolla Cancer Research Foundation | Inhibition of transforming growth factor beta activity |
| US5654270A (en) * | 1988-06-28 | 1997-08-05 | La Jolla Cancer Research Foundation | Use of fibromodulin to prevent or reduce dermal scarring |
| ATE233568T1 (en) * | 1991-11-14 | 2003-03-15 | Jolla Cancer Res Found | INHIBITORS OF CELL REGULATORY FACTORS AND METHODS FOR PREVENTING OR REDUCING SCARING |
| US5830847A (en) * | 1992-10-30 | 1998-11-03 | Hsc Research & Development Limited Partnership | Soluble TGF-β-binding endoglin polypeptides and homodimers |
| US5650389A (en) * | 1993-03-01 | 1997-07-22 | University Of Alabama At Birmingham Research Foundation | Methods for the inhibition of complement activation |
| US5453492A (en) * | 1993-07-28 | 1995-09-26 | La Jolla Cancer Research Foundation | 60 kDa transforming growth factor-β-binding protein and its use to detect or purify TGF-β |
| US5567807A (en) * | 1994-07-08 | 1996-10-22 | La Jolla Cancer Research Foundation | Processes for the purification of human recombinant decorin and the detection of guanidinium ions |
| WO1999021965A2 (en) | 1997-10-28 | 1999-05-06 | President And Fellows Of Harvard College | In vitro differentiation of vascular smooth muscle cells, methods and reagents related thereto |
| WO2001085192A1 (en) * | 2000-05-05 | 2001-11-15 | Gtc Biotherapeutics, Inc. | Transgenically produced decorin |
| IL160983A0 (en) | 2001-09-24 | 2004-08-31 | Imp College Innovations Ltd | Use of pyy for preparation of a medicament for modification of feeding behavior |
| AU2003201998C1 (en) | 2002-01-10 | 2012-10-25 | Imperial Innovations Limited | Modification of feeding behavior |
| US20030180301A1 (en) * | 2002-01-22 | 2003-09-25 | Shaf Keshavjee | Use of TGF-beta antagonists to treat or to prevent chronic transplant rejection |
| JP5128935B2 (en) * | 2004-03-31 | 2013-01-23 | ジェネンテック， インコーポレイテッド | Humanized anti-TGF-β antibody |
| WO2006044433A2 (en) * | 2004-10-13 | 2006-04-27 | The Ohio State University Research Foundation | Methods to treat or prevent viral-associated lymphoproliferative disorders |
| KR100747646B1 (en) * | 2005-02-25 | 2007-08-08 | 연세대학교 산학협력단 | Gene Delivery System Containing Decorin Gene and Pharmaceutical Composition for Treating Cancer containing the System |
| JP2006295872A (en) * | 2005-03-18 | 2006-10-26 | Matsushita Electric Ind Co Ltd | Formation method of device-dependent key, confidential information lsi equipped with secret information function using the method, host equipment using the lsi, record medium with authentication function used for the host equipment, and mobile terminal with recording medium equipped with authentication function |
| EP1940842B1 (en) | 2005-09-29 | 2012-05-30 | Merck Sharp & Dohme Corp. | Acylated spiropiperidine derivatives as melanocortin-4 receptor modulators |
| EP1948675B1 (en) | 2005-10-25 | 2014-07-30 | The Johns Hopkins University | Methods and compositions for the treatment of marfan syndrome and associated disorders |
| US8454952B2 (en) | 2006-03-13 | 2013-06-04 | The Johns Hopkins University | Augmentation of endothelial thromboresistance |
| EP2083831B1 (en) | 2006-09-22 | 2013-12-25 | Merck Sharp & Dohme Corp. | Method of treatment using fatty acid synthesis inhibitors |
| EP2083863B1 (en) | 2006-10-03 | 2015-03-18 | Genzyme Corporation | Antibodies to tgf-beta for use in the treatment of infants at risk of developing bronchopulmonary dysplasia |
| US9053063B2 (en) | 2007-02-21 | 2015-06-09 | At&T Intellectual Property I, Lp | Method and apparatus for authenticating a communication device |
| EP2145884B1 (en) | 2007-04-02 | 2014-08-06 | Msd K.K. | Indoledione derivative |
| LT3117709T (en) | 2010-03-12 | 2018-11-26 | Genzyme Corporation | Combination therapy for treating breast cancer |
| US8956868B2 (en) | 2010-12-27 | 2015-02-17 | Lsip, Llc | Induced pluripotent stem cells produced with a connexin inhibitor and a TGF-β signaling inhibitor |
| WO2012125626A2 (en) * | 2011-03-14 | 2012-09-20 | Catalent Pharma Solutions Llc | Decorin compositions and use thereof |
| WO2013014262A1 (en) | 2011-07-27 | 2013-01-31 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Methods for diagnosing and treating myhre syndrome |
| WO2013062544A1 (en) | 2011-10-26 | 2013-05-02 | Seattle Children's Research Institute | Cysteamine in the treatment of fibrotic disease |
| JP6218229B2 (en) | 2011-12-28 | 2017-10-25 | 京都府公立大学法人 | Normalization of corneal endothelial cell culture |
| WO2014176198A1 (en) | 2013-04-22 | 2014-10-30 | Catalent Pharma Solutions, Llc | Veterinary decorin compositions and use thereof |
| JPWO2015064768A1 (en) | 2013-10-31 | 2017-03-09 | 京都府公立大学法人 | Drugs for diseases related to endoplasmic reticulum cell death in corneal endothelium |
| US10882903B2 (en) | 2015-05-18 | 2021-01-05 | Arizona Board Of Regents On Behalf Of The University Of Arizona | Methods and compositions for treating an alphavirus infection |
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| US5583103A (en) * | 1988-06-28 | 1996-12-10 | La Jolla Cancer Research Foundation | Inhibition of transforming growth factor beta activity |
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| JP2003034651A (en) | 2003-02-07 |
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| US5726149A (en) | 1998-03-10 |
| EP0349480A1 (en) | 1990-01-03 |
| FI906411A0 (en) | 1990-12-27 |
| DE68921803T2 (en) | 1995-07-13 |
| AU633011B2 (en) | 1993-01-21 |
| JP2887325B2 (en) | 1999-04-26 |
| DE68921803D1 (en) | 1995-04-27 |
| ATE120235T1 (en) | 1995-04-15 |
| JPH09117278A (en) | 1997-05-06 |
| JPH03503846A (en) | 1991-08-29 |
| DK306890A (en) | 1990-12-28 |
| US6046162A (en) | 2000-04-04 |
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